Bile acids salts which are organic acids derived from cholesterol are natural ionic detergents that play a pivotal role in the absorption, transport, and secretion of lipids. In bile acid chemistry, the steroid nucleus of bile acids salts has the perhydrocyclopentano phenanthrene nucleus common to all perhydrosteroids. Distinguishing characteristics of bile acids include a saturated 19-carbon sterol nucleus, a beta-oriented hydrogen at position 5, a branched, saturated 5-carbon side chain terminating in a carboxylic acid, and an alpha-oriented hydroxyl group in the 3-position. The only substituent occurring in most natural bile acids is the hydroxyl group In most mammals the hydroxyl groups are at the 3, 6, 7 or 12 positions.
The common bile acids differ primarily in the number and orientation of hydroxyl groups on the sterol ring. The term, primary bile acid refers to these synthesized de novo by the liver. In humans, the primary bile acids include cholic acid (3.alpha., 7.alpha., 12.alpha.-trihydroxy-5.beta.-cholanic acid) ("CA") and chenodeoxycholic acid (3.alpha., 7.alpha.-dihydroxy-5.beta.-cholanic acid) ("CDCA"). Dehydroxylation of these bile acids by intestinal bacteria produces the more hydrophobic secondary bile acids, deoxycholic acid (3.alpha., 12.alpha.-dihydroxy-5.beta.-cholanic acid) ("DCA") and lithocholic acid (3.alpha.-hydroxy-5.beta.-cholanic acid) ("LCA"). These four bile acids CA, CDCA, DCA, and LCA, generally constitute greater than 99 percent of the bile salt pool in humans. Secondary bile acids that have been metabolized by the liver are sometimes denoted as tertiary bile acids.
Keto-bile acids are produced secondarily in humans as a consequence of oxidation of bile acid hydroxyl groups, particularly the 7-hydroxyl group, by colonic bacteria. However, keto-bile acids are rapidly reduced by the liver to the corresponding .alpha. or .beta.-hydroxy bile acids. For example, the corresponding keto bile acid of a CDCA is 7-keto lithocholic acid and one of its reduction products with the corresponding .beta.-hydroxy bile acid is ursodeoxycholic acid (3.alpha.-7.beta.-dihydroxy-5.beta.-cholanic acid) ("UDCA"), a tertiary bile acid.
UDCA, a major component of bear bile, has been used for the treatment of and the protection against many types of liver disease for a little over 70 years as a major pharmaceutical agent. Its medicinal uses include the dissolution of radiolucent gall stones, the treatment of biliary dyspepsias, primarily biliary cirrhosis, primary sclerosing choplangitis, chronic active hepatitis and hepatitis C. In other mammalian species, bile acids containing a 6.beta.-hydroxyl group, which are found in rats and mice, are known as muricholic acid; 6.alpha.-hydroxy bile acids produced by swine are termed hyocholic acid and hyodeoxycholic acids. 23-hydroxy bile acids of aquatic mammals are known as phocecholic and phocedeoxycholic acids.
Under normal circumstances, more than 99 percent of naturally occurring bile salts secreted into human bile are conjugated. Conjugates are bile acids in which a second organic substituent (e.g. glycine, taurine, glucuronate, sulfate or, rarely, other substituents) is attached to the side chain carboxylic acid or to one of the ring hydroxyl groups via an ester, ether, or amide linkage. Therefore, the ionization properties of conjugated bile acids with glycine or taurine are determined by the acidity of the glycine or taurine substituent.
Free, unconjugated, bile acid monomers have pKa values of approximately 5.0. However, pKa values of glycine conjugated bile acids are on average 3.9, and the pKa of taurine conjugate bile acids are less than 1.0. The effect of conjugation, therefore, is to reduce the pKa of a bile acid so that a large fraction is ionized at any given pH. Since the ionized salt form is more water soluble than the protonated acid form, conjugation enhances solubility at a low pH. Free bile acid salts precipitate from aqueous solution at pH 6.5 to 7. In contrast, precipitation of glycine conjugated bile acid occurs only at pH of less than 5. Taurine conjugated bile acids remain in aqueous solution under very strongly acidic conditions (lower than pH 1). However, in the gastric pH range, certain bile acids such as UDCA and CDCA are no longer soluble.
Conjugation of the side chain of a bile acid with glycine or taurine has little influence on the hydrophobic activity of fully ionized bile salts. More hydrophobic bile salts exhibit greater solubilizing capacity for phospholipid and cholesterol and are consequently better detergents. More hydrophobic bile salts are also more injurious to various membranes, both in vivo and in vitro.
Natural bile salt pools invariably contain multiple bile acid salts. Mixtures of two or more bile salts of differing hydrophobic activity may behave as a single bile salt of an intermediate hydrophobic activity. As a result, detergent properties and the toxicity of mixtures of two bile acids of differing hydrophobic activity often are intermediate between the individual components. Biologic functions and biologic properties of bile acids resulting from their amphiphillic properties are as follows:
I. Bile acid synthesis from cholesterol is one of the two principal pathways for the elimination of cholesterol from the body. PA1 II. Bile flow is generated by the flux of bile salts passing through the liver. Bile formation represents an important pathway for solubilization and excretion of organic compounds, such as bilirubin, endogenous metabolites, such as emphipathic derivatives of steroid hormones; and a variety of drugs and other xenobiotics. PA1 III. Secretion of bile salts into the bile is coupled with the secretion of two other biliary lipids, that is, phosphatidylcholine (lecithin) and cholesterol; the coupling of bile salt output with the lecithin and cholesterol output provides a major pathway for the elimination of hepatic cholesterol. PA1 IV. Bile salts, along with lecithin, solubilize cholesterol in bile in the form of mixed micelles and vesicles. Bile salt deficiency, and consequently reduced cholesterol solubility in bile, may play a role in the pathogenesis of cholesterol gallstones. PA1 V. Bile acids are thought to be a factor in the regulation of cholesterol synthesis. At present, it is not certain whether they regulate the cholesterol synthesis by acting directly on the hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase or indirectly by modulating the cholesterol absorption in the intestine. PA1 VI. Bile salts in the enterohepatic circulation are thought to regulate the bile acid synthesis by suppressing or derepressing the activity of cholesterol 7-hydroxylase, which is the rate-limiting enzyme in the bile acid biosynthesis pathway. PA1 VII. Bile acids may play a role in the regulation of hepatic lipoprotein receptors (apo B.E.) and consequently may modulate the rate of uptake of lipoprotein cholesterol by the liver. PA1 VIII. In the intestines, bile salts in the form of mixed micelles participate in the intraliminal solubilization, transport, and absorption of cholesterol, fat-soluble vitamins, and other lipids. PA1 IX. Bile salts may be involved in the transport of calcium and iron from the intestinal lumen to the brush border. PA1 improvement of the oral absorption of an intrinsically, biologically active, but poorly absorbed hydrophillic and hydrophobic drug; liver site-directed delivery of a drug to bring about high therapeutic concentrations in the diseased liver with the minimization of general toxic reactions elsewhere in the body; and gallbladder-site delivery systems of cholecystographic agents and cholesterol gallstone dissolution accelerators. As an example, in 1985, Drs. Gordon & Moses et al. demonstrated that therapeutically useful amount of insulin are absorbed by the nasal mucosa of human beings when administered as a nasal spray with common bile salts such as DCA, UDCA, CDCA, CA, TUDCA, TCDCA, etc. See Moses, Alan C., et al., Diabetes vol. 32 (November 1983) 1040-1047; Gordon, G. S., et al., Proc. Nat'l Acad. Sci. USA, vol. 82 (November 1985) 7419-7423. In their experiment, bile acids produced marked elevations in serum insulin concentration, and about 50 percent decreases in blood glucose concentrations. However, this revolutionary nasal spray solution dosage form with bile acids (salts) as a adjuvant could not be developed further and commercialized, because the nasal spray solution must be prepared immediately prior to use due to the precipitation of bile acid salt and the instability of insulin at pH levels between 7.4 and 7.8. Moreover, as indicated in this disclosure, ursodeoxycholic acid as an adjuvant could not be used because of its insolubility at pH between 7.4 and 7.8.
Recent drug delivery research concerning the characteristics and biofunctions of naturally occurring bile acid as an adjuvant and/or a carrier has focused on the derivatives and analogs of bile acids and bile acids themselves as novel drug delivery systems for delivery to the intestinal tract and the liver. These systems exploit the active transport mechanism to deliver aimed drug molecules to the specific target tissue by oral or cystic administration. Thus, if bile acids or bile acid derivatives are rapidly and efficiently absorbed in the liver and, consequently, undergo enterohepatic cycling, many potential therapeutic applications are foreseen including the following:
The pH of the commercial insulin injection solutions are between 2.5 and 3.5 for acidified dosage forms and is between 7.00 and 7.4 for neutral dosage forms. Therefore, the safe and efficient preparations of any solution dosage forms of insulin with bile acid (salt) are not commercially available at this time, because of physically chemically incompatible characteristics of bile acids salts insolubility and the stability of insulin in acidic and neutral pH.
Heparin, a most potent anticoagulant, is widely used in the treatment of and in the prevention of thromboembolism. However, heparin treatment is usually limited to hospitalized patients since this drug is given only by injection. Alternate routes which have been attempted are an intrapulmonary spray, suppositories, and enema. According to numerous publications, for heparin absorption through the gastrointestinal mucosa to be facilitated, the preparations should be in acidic condition. According to Dr. Ziv, Dr. Eldor et al., heparin was absorbed through the rectal mucosa of rodents and primates only when administered in solutions containing sodium cholate or sodium deoxycholate. See Ziy E. et al., Biochemical Pharmacology, vol. 32, No. 5, pp. 773-776 (1983). Unfortunately, heparin is only stable in acidic conditions. Bile acids are particularly not soluble in acidic conditions. Therefore, due to their incompatible characteristics, the commercial dosage forms which heparin can be absorbed through the gastrointestinal mucosa with bile acids (salts) are not available at this time.
Drug delivery systems involving bile acids can provide liver-specific drug targeting which is of major interest for drug development since standard pharmacological approaches to liver diseases have been frustrated by the inadequate delivery of active agents into liver cells as well as non specific toxicity towards other organs. For example, the liver-specific delivery of a drug is necessary for inhibitors of collagen synthesis for the treatment for liver fibrosis in order to avoid unspecific and undesired side-effects in extrrhepatic tissues. Furthermore, for the treatment of cancer of the biliary system, high drug levels must be achieved in the liver and the biliary system, whereas in extrahepatic tissues low drug concentrations are desired to minimize the cytoxicity of the cytostatics to normal non-tumor cells. Dr. Kramer, Dr. Wess et al. demonstrate that hybrid molecules formed by covalent linkages of a drug to a modified bile acid molecule are recognized by the Na+-dependent bile acid uptake systems in the liver and the ileum. See U.S. Pat. No. 5,641,767. Even if bile acid salts and their derivatives act as shuttles for specific delivery of a drug to the liver, as already mentioned above, there are enormous risks to the development of the derivatives of bile acids or bile acid salts as carriers because new derivatives of bile acids or bile acid salts formed by covalent linkages of a drug to bile acid must be tested for its pharmacology, toxicity and clinical effectiveness. Thus, the development of preparations in which a drug can be absorbed with bile acids or bile acid salts from the places which contain the excessive bile acids in the intestine is far easier and far more valuable than the development of the new bile acid derivatives because less testing is required.
In spite of the extremely valuable therapeutic activities and the long historic medical uses of bile acids as therapeutically active agents and as carriers and/or adjuvants based on the already mentioned biological properties and functions of bile acids, the commercial administration of bile acids are limited to the pharmaceutical formulations with a solid form of bile acid which are in tablet, capsule and suspension because of its insolubility to aqueous media at pH from approximately 1 to 8, and its extremely bitter taste and equally bitter after-taste which lasts several hours. Note that ursodeoxycholic acid, chenodeoxycholic acid, and lithocholic acid are practically insoluble in water; that deoxycholic acid and cholic acid have solubilities of 0.24 g/l, and 0.2 g/l, respectively, and that tauroursodeoxycholic acid, taurochenodeoxycholic acid, and taurocholic acid are insoluble in hydrochloric acid solution. The few aqueous dosage forms that are available are unstable, and have very limited uses because of pH control and maintenance problems. Moreover, some commercial pharmaceutical dosage forms of bile acids have been shown to have scant bioavailability as described in European Journal of Clinical Investigation (1985) 15, 171-178. Bile acid, especially ursodeoxycholic acid is poorly soluble in the gastro-duodeno jejunal contents of fasted subjects. From 21% to 50% of the ingested doses were recovered in solid form because of the unpredictable variations in the very slow progressive solubilization of solid ursodeoxycholic acid in the gastrointestinal track. Bile acids, particularly ursodeoxycholic acid, deoxycholic acid, chenodeoxycholic acid, cholic acid, hyodeoxycholic acid, 7-keto lithocholic acid, tauroursodeoxycholic acid, and taurochenodeoxycholic acid among others, are especially insoluble in the gastric juices and in aqueous hydrochloric acid solution. However, the solubility of bile acids increase with the increase of the pH in the intestine very slowly and incompletely, and eventually the bile acids become soluble at pH between 8 and 9.5.
To overcome this slow and inefficient absorption process in the intestine due to the incomplete and slow solubilization of bile acids, many newly developed pharmaceutical formulations have been prepared, such as delayed release dosage forms with water soluble solid bile acids which are often strongly alkaline. These newly developed pharmaceutical dosage forms are enterosoluble-gastro resistant. These enterosoluble-gastroresistant dosage forms remain intact in gastric juices in the stomach, but are dissolved and release the strongly alkaline solid bile salts of the formulations at the targeted area, within a limited time once they reach the small intestine.
These types of dosage forms, of course, showed better bioavailability than presently commercialized dosage forms as described in U.S. Pat. No. 5,380,533. However, it is extremely difficult and very costly to prepare the precise delayed release dosage forms which can release therapeutically active components by disintegration, dissolution and diffusion at the desired area within a limited time. According to U.S. Pat. No. 5,302,398, the absorption test of the gastroresistant enterosoluble dosage forms of bile acids, particularly ursodeoxycholic acid in man show that its absorption increases a value of about 40 percent in comparison with administering the same amount in current commercial dosage forms. Its maximum hematic concentrations are on average three times higher, and are reached faster than with the commercial formulations. Any dosage forms of bile acid formula must be capable of releasing bile acids in a known and consistent manner following administration to the patient. Both the rate and the extent of release are important, and should be reproducible. Ideally, the extent of release should approach 100 percent, while the rate of release should reflect the desired properties of the dosage form.
It is a well-known fact that solution dosage forms of drugs show significantly improved rates and extents of absorption, compared to the same drug formulated as a tablet, capsule, or suspension. This is because solution dosage forms are chemically and physically homogeneous solutions of two or more substances. Moreover, the specially designed solution dosage forms which can maintain the solution systems without breaking down under any pH conditions are ready to be diffused in the desired area for immediate and complete absorption, whereas tablets, capsules or delayed release formulations must invariably undergo disintegration, dissolution and diffusion at the desired area within a limited time. Once again, unpredictable variations in the extent and rate of release of bile acids by the disintegration, dissolution and diffusion of delayed or immediate release dosage forms having pH-dependent instability result in the slow and inefficient absorption, and reduced bioavailability in comparison with the solution dosage forms which can reach the targeted area throughout the gastrointestinal track without any break-down of the solution system caused by the pH of the environment in the stomach and intestines. When the therapeutically active ingredients in aqueous solution forms are not precipitated as solid by acidic gastric juices in the stomach and by the various alkaline pH levels of the intestine, the formulation overcomes as a natural consequence, the scarce bioavailability resulted by the unexpected, undesirable results for the extent and the rate of release by disintegration, dissolution and/or diffusion should be overcome.